Gene encoding desulfurases

Details Gene encoding desulfurases Gene encoding desulfurases

2000-09-29

2002-07-16

Saidha, Tekchand (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Lyase

C435S282000, C435S252300, C435S320100, C536S023200

Reexamination Certificate

active

06420158

ABSTRACT:

TECHNICAL FIELD
The present invention relates to enzymes having the function of decomposing, using microorganisms, thiophene compounds, namely benzothiophene, dibenzothiophene (hereinafter referred to as “DBT”) and their substituted compounds, or derivatives thereof, and genes encoding the enzymes. By using the enzymes and the gene defined in the present invention, sulfur can be released from benzothiophene, DBT and their substituted compounds, or derivatives thereof which are contained in fossil fuels such as petroleum. As a result, sulfur, which is generally diffused in the air when fossil fuels such as petroleum and coal are burned, can be easily removed from the fossil fuel.
PRIOR ART
In order to remove sulfur from hydrocarbon fuel such as petroleum, methods including alkali treating or solvent desulfurization are known. However, at present, mainly hydrodesulfurization is used. Hydrodesulfurization is a method for reacting sulfur compounds in a petroleum fraction with hydrogen in the presence of a catalyst and removing the produced hydrogen sulfide so as to obtain low-sulfur products. As a catalyst, metallic catalysts such as cobalt, molybdenum, nickel and tungsten are used with alumina as a carrier. When the molybdenum on alumina is used as the catalyst, usually cobalt or nickel is added as a promoter to enhance catalysis performance. The hydrodesulfurization with metallic catalysts is undoubtedly a fine process which is widely used throughout the world at the moment. However, as a process for producing petroleum products adapted to more strict environmental regulations, there are some problems. Some examples are discussed below briefly.
Generally the substrate specificity of a metallic catalyst is low, and so it is suitable for decomposing various kinds of sulfur compounds and lowering the amount of sulfur contained in the fossil fuel as a whole. However, it is considered that the effect of desulfurization with metallic catalyst is sometimes insufficient for a specific group of sulfur compounds, i.e., heterocyclic sulfur compounds such as benzothiophene, DBT and their alkyl derivatives. For example, after desulfurizing light oil, various heterocyclic organic sulfur compounds still remain. One reason why the effect of desulfurization with metallic catalyst is insufficient would be steric hindrance caused by substituents which are around the sulfur atoms of the organic sulfur compounds. Among these substituted compounds, the influence of a methyl substituted compound on the reaction of a metallic catalyst has been studied in relation to thiophene, benzothiophene, DBT and so on. According to such studies, it is generally said that, as the number of substituted compounds increases, desulfurization reaction rates decreases. However, it is also said that the position of the substituents have a very large influence on the reactivity. One of the reports which have shown that the steric hindrance has the significant influence on the reaction of metallic catalyst is, for example, Houalla, M., Broderick, D. H., Sapre, A. V., Nag, N. K., de Beer, V. H., Gates, B. C., Kwart, H. J., Catalt., 61, 523-527(1980). In fact, it is known that a considerable amount of various alkyl derivatives of DBT exists in light oil (e.g. Kabe, T., Ishihara, A. and Tajima, H. Ind. Eng. Chem. Res., 31, 1577-1580(1992)).
As stated above, it is considered that, in order to desulfurize organic sulfur compounds which are resistant against hydrodesulfurization, higher reaction temperature and pressure than that usually used are required, and also the amount of hydrogen added to be increased remarkably. It is thus expected that enormous capital investment and operating costs are needed to improve a hydrodesulfurization process such as this. For example, light oil contains organic sulfur compounds resisting such hydrodesulfurization as a major compound species, and as stated above, a substantial improvement on the hydrodesulfurization process is required to carry out more sophisticated desulfurization of light oil (an ultra deep desulfurization).
On the other hand, the enzyme-reaction in an organism proceeds under relatively mild conditions, and further, the rate of enzyme reaction in an organism compares favorably with that of a chemical catalyst. Moreover, there are so many kinds of enzymes in vivo to conform appropriately to various kinds of vital reactions occurring therein, and those enzymes usually show a very high substrate specificity. These characteristics are expected to be utilized for so-called biodesulfurization reaction, which removes sulfur from sulfur compounds in fossil fuel by using microorganisms (Monticello, D. J., Hydrocarbon Processing 39-45(1994)).
There are a large number of reports on methods for removing sulfur from heterocyclic sulfur compounds which are ingredients of petroleum by using bacteria, and these methods are broadly divided into the reaction of decomposing a ring (C—C bond cleavage) and the C—S bond cleavage reaction. As bacteria having C—C-bond-attacking desulfurization activity, for example, strains belonging to Pseudomonas sp.,
Pseudomonas aeruginosa,
Beijerinckia sp.,
Pseudomonas alcaligenes, Pseudomonas stutzeri, Pseudomonas putida,
Brevibacterium sp. are known. These bacteria carry out the cleavage of C—C bond in heterocyclic sulfur compounds of which a representative example is DBT, decompose a benzene ring, thereafter, by oxidative reaction cascade, they conduct a metabolism in which salt containing sulfur atom(s) is released. As the reaction mechanism of the carbon-backbone-attacking pathway, there are the hydroxylation of aromatic ring (DBT→→1,2-dihydroxyDBT), the cleavage of a ring, and the oxidation to water-soluble product (1,2-dihydroxy DBT→→trans-4 [2-(3-hydroxy)thianaphthenyl]-2-oxo-butenoic acid, 3-hydroxy-2-formylbenzothiophene), and this reaction mechanism is called “Kodama pathway”. The C—C bond in a benzene ring of DBT is attacked by this kind of reaction to generate various water-soluble substances which are extractable from the oil. Due to this reaction, however, other aromatic molecules in the oil are also attacked, and as a result, a significant amount of hydrocarbons move to water phase (Hartdegen, F. J., Coburn, J. M. and Roberts, R. L. Chem. Eng. Progress, 80, 63-67(1984)). This causes the reduction of total calories of petroleum and so it is an industrially ineffective reaction. Furthermore, as Kodama et al. has reported, this type of bacteria oxidatively decomposing DBT provides water-soluble thiophene compounds (mainly 3-hydroxy-2-formylbensothiophene) as oxidized products, but this is a substance difficult to remove from water phase. In addition, since the attack to the carbon ring of DBT often occurs at position 2 or 3 of DBT, DBT substituted with an alkyl or alkyl groups at these positions does not become the substrate of the Kodama pathway.
It has been reported that there are microorganisms which decompose not only crude oil or coal but also model compounds containing sulfur, remove. selectively hetero-atom sulfur, and generate sulfate and hydroxyl compounds. Taking the structure of the metabolites into consideration, this kind of reaction is considered to be one which cleaves specifically C—S bond in sulfur compounds and accordingly releases sulfur in the form of sulfate. As shown in Table 1, to date, some biodesulfurization reaction systems which are characterized by attacking sulfur have been reported.
TABLE 1
C—S bond attacking bacteria
STRAIN
SUBSTRATE
DECOMPOSED PRODUCT
REFERENCE DOCUMENTS
Pseudomonas sp. CB1
dibenzothiophene; coal
hydroxybiphenyl + sulfate
Isbister et al. (1985)
Acinetobacter sp. CB2
dibenzothiophene
hydroxybiphenyl + sulfate
Isbister et al. (1985)
Gram-positive bacteria
coal
sulfate
Crwaford et al. (1990)
Rhodococcus rhodochrous
IGTS8
dibenzothiophene
hydroxybiphenyl + sulfate
Kilbane (1989)
(ATCC 53968)
coal; petroleum
Desulfovibrio desulfuricans
dibenzothiophene
biphenyl + hydrogen sulfide
Kim et al. (1990)
Corynebacterium sp.
dibenzothiophene
hydroxybiphenyl

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